The complement system is an integral part of the innate immunity and contributes to the recognition and elimination of pathogens, clearance of apoptotic cells or immune complexes and modulation of the adaptive immune response (Ricklin et al. 2010, Carroll et al. 2011). Complement is composed of a number of plasma proteins produced mainly by the liver, normally circulating as zymogens or as membrane proteins and operates in plasma, in tissues, or within cells. The activation of the three complement pathways the classical (CP), the lectin (LP) or the alternative pathway (AP) each results in the formation of C3 convertases (C4b2a in the CP, LP or C3bBb in the AP) which catalyzes the cleavage of the central component of the complement system, C3, into the activation product C3b and the anaphylactic peptide C3a. Subsequently and in a cascade-like triggered reaction, pathogens are marked for destruction and a series of inflammatory responses is induced recruiting immune cells to fight infection and maintain homeostasis (reviewed in (Merle et al. 2015)). Both, inefficient activation or inefficient regulation of complement may be causative for or contribute to a number of infections or non-infectious diseases, including immunodeficiency, autoimmunity, chronic inflammation, thrombotic microangiopathy, graft rejection as well as renal and retinal diseases such as atypical hemolytic uremic syndrome (aHUS), C3 Glomerulopathies (C3G) and age-related macular degeneration (AMD) (Holers 2008).
The Alternative Pathway (AP)
While the CP and LP are triggered by certain recognition molecules, the AP is permanently active at a low level. This occurs by a mechanism called “tick-over”, that is initiated by hydrolysis of the internal C3 thioester of the C3b-like molecule C3(H2O) to form its bioactive form. Together with the soluble Factor B (FB) and Factor D (FD), that cleaves C3(H20) bound FB, fluid phase C3 convertase complexes (C3b(H20)Bb) are generated and native C3 molecules are cleaved and activated. Activated C3b binds covalently, via a thioester-containing domain (TED or C3d domain), to hydroxyl groups of any adjacent surfaces. On pathogens, C3b in the immediate proximity to the site of its generation is accumulated and further C3 convertases (C3bBb) are formed, thereby amplifying complement activation. The binding of a second C3b to the C3 convertase leads to the generation of the C5 convertase (C3bBbC3b) that initiates the cleavage of C5 into the potent immune effector molecule C5a and C5b. C5b recruits complement components C6, C7, C8 and C9 forming the C5b-9 terminal membrane attack complex (MAC) which results in lysis of pathogens (Ricklin et al. 2010, Carroll et al. 2011).
Regulation of the AP
The AP has to be precisely regulated to allow rapid elimination of opsonized cells and pathogens and to minimize unrestricted AP activation that may cause host tissue damage. Healthy host cells are usually protected from complement mediated attack by a number of membrane-bound or soluble proteins of the regulator of complement activation (RCA) family that act on different activation levels of the cascade, some of them having overlapping functionality, while others have unique complement regulatory properties (Zipfel et al. 2009).
The generation of new C3b is strictly controlled by RCA proteins acting as cofactors for Factor I (FI) mediated irreversible degradation of C3b into iC3b or by destabilization of the C3bBb convertase complexes (Decay Acceleration Activity, DAA). In addition, RCAs can interfere with C5 convertase activity, thereby controlling the cleavage of C5 into its activation products C5a and C5b or by factors binding to terminal complement complex (TCC) compounds thereby preventing insertion of the MAC complex in the membrane. Together with membrane cofactor protein (MCP or CD46), complement receptor 1 (CR1 or CD35), decay accelerating factor (DAF or CD55) membrane inhibitor of reactive lysis (MIRL, CD59) and the soluble factors vitronectin and clusterin and others, members of the Factor H/FHR protein family, in particular FH, supports regulation of complement in circulation and on surfaces to which it specifically binds.
The Factor H/FHR Protein Family
The factor H/FHR protein family comprises a group of highly related plasma proteins that includes the five complement Factor H-related proteins (FHRs), FHR1, FHR2, FHR3, FHR4, FHR5, Factor H (FH) and the spliced variant Factor H-like protein 1 (FHL-1). Each single gene of the family members is located on a distinct segment on human chromosome 1q32 within the RCA gene cluster (Skerka et al. 2013). While FH is the main regulator of the alternative pathway, the functions of the FHRs are not completely understood. It has been suggested that the interaction of FHR proteins modulate the complement regulatory activity on cell surfaces (Jozsi et al. 2015). In addition, their ability for homo- and hetero-oligomerization can increase the avidity of FHR1, FHR2 and FHR5 for their ligands. This has been proposed as a fine tuning like mechanism in the recognition and modulation of complement activation (Jozsi et al. 2015). However, some FH independent and unique complement regulatory properties have also been described for FHR1 and FHR2. Since FH, FHR1 and FHR2 can downregulate complement activation, they have been proposed as promising candidates to modulate complement activation under pathological conditions (Licht et al. 2005, Licht et al. 2006, Skerka et al. 2013, Haffner et al. 2015).
Among the FH/FHR protein family, FH is the most abundant complement protein circulating in plasma at concentrations of ˜350-600 μg/ml. With a molecular weight of 155 kDa, the monomeric glycoprotein FH regulates the AP and the amplification loop of the complement pathways. FH consists of 20 repetitive short consensus repeat (SCR) domains and regulates the activation of C3 convertases in fluid phase as well as on cell surfaces. The N-terminal domains SCR1-4 contain the complement regulatory region of the protein. FH SCR1-4 binds C3b and thereby prevents the formation of the C3 and C5 convertases and facilitates the disassembly of already formed convertases by competing with FB for C3b binding (Weiler et al. 1976). Additionally, these domains are relevant for FH to act as a cofactor for FI mediated C3b inactivation (Gordon et al. 1995, Rodriguez de Cordoba et al. 2004, Alexander et al. 2007, de Cordoba et al. 2008). The C-terminus of FH (SCR 19-20) primarily represents the binding recognition domain that interacts with C3b, C3d, pentraxins, extracellular matrix and cellular surfaces (Jarva et al. 1999, Oppermann et al. 2006, Hebecker et al. 2013). Binding of FH on cell surfaces or biological membranes is mediated by polyanionic structures like glycosaminoglycans (GAG) (e.g. heparin) or sialic acids, and regulates local complement activation on endogenous cells, such as glomerular endothelial cells or the glomerular basement membrane (GBM) (Jozsi et al. 2004, Ferreira et al. 2006, Jozsi et al. 2007, Blaum et al. 2015).
FHR1 is composed out of five SCRs (Skerka et al. 1991) and has two isoforms. Two glycosylated forms (FHR1α ˜41 and FHR1β ˜37 kDa) with either one or two carbohydrate side chains circulate in human plasma with a concentration of about 100 μg/ml. FHR1 has a high C-terminal sequence homology to FH and C-terminal SCR1 and SCR2 have high amino acid identity to SCR1 and SCR2 of FHR2 (97% and 100%, respectively) and to SCR1 and SCR2 of FHR5 (91% and 83%, respectively). FHR1 regulates C5-convertase activity and inhibits complement activation while C3-convertase activity is uninfluenced. N-terminal SCR1-2 binds to C5 and C5b6, whereas the C-terminal SCR3-5 binds to C3b, C3d and heparin. Supposedly, FHR inhibits C5 activation and cleavage into C5a and C5b by binding of SCR1-2 to C5. In addition FHR1 is a terminal pathway regulator and inhibits the assembly of the MAC, presumably by binding of SCR1-2 to C5b6 complex (Heinen et al. 2009).
FHR2 consists of four SCRs (Skerka et al. 1992) displaying amino acid identity to SCR 6-7 and 19-20 of FH. The N-terminal SCR1 of FHR2 is almost identical to FHR1 and FHR5 and allows the formation of homodimers and heterodimers with FHR1, but not with FHR5. FHR2 circulates in the human plasma at concentrations of about 50 μg/ml. FHR2 regulates complement activation, presumably via a mechanism in which FHR2 bound C3 convertases do not cleave the substrate C3. Interestingly, FHR2 hardly competes off factor H from C3b. FHR2 does not compete with FH for binding to C3b at physiological concentrations (Goicoechea de Jorge et al. 2013).
FHR1 like FHR2 and FHR5 each contain a conserved dimerization interface built up of residues Tyr34, Ser36 and Tyr39 located in SCR1 that play a key role in the assembling and facilitates homo- and heterodimer formation transmitted by a tight antiparallel binding of the N-terminus. In addition to its regulatory functions, FHRs prevent FH binding (or compete off FH) to C3b or host/pathogen cell surfaces under certain conditions causing a deregulation (activation) of complement (Jozsi et al. 2015). The formation of multimeric FHR complexes might increase local concentration thereby increasing avidity and/or affinity toward its substrate or toward surfaces that are to be regulated by FHRs. It has been suggested that deregulation may be intensified under pathophysiological conditions for example due to abnormal multimerisation of FHRs in C3 Glomerulopathies which enhances ligand binding and FH competition (Goicoechea de Jorge et al. 2013).
AP Associated Diseases
Dysregulation of the AP caused by mutations, dysfunctional polymorphisms in complement components and regulators such as FH or antibodies that promote activation of the AP are highly associated with diseases such as atypical hemolytic uremic syndrome (aHUS) (Noris et al. 2009) or C3 glomerulopathies (C3G) (Barbour et al. 2013) age-related macular degeneration (AMD) (Kawa et al. 2014) or paroxysomal nocturnal hemoglobinuria (PNH) (Holers 2008). Beside these a pathogenic role for the AP has been shown or postulated for IgA-nephropathy (Maillard et al. 2015), systemic lupus erythematosus—(SLE) (Wilson et al. 1976), ischemia-reperfusion (IR) damage or transplant rejection, rheumatoid arthritis (RA) and many others (Holers 2008) (Ricklin et al. 2013).
Atypical HUS and C3G are master models for AP related disease. In aHUS, mutations in either components of the AP or its regulators (C3, FB, FI, FH, FHR1 or MCP) or anti-FH antibodies lead to uncontrolled complement activation and ultimately formation of C5b-9 and endothelial cell damage. This is accompanied by glomerular thrombotic microangiopathy and acute renal failure, historically resulting in death or terminal renal failure in more than 60% of the patients. In C3G, which progresses to terminal renal failure in more than 50% of the patients within ten years, complement deposits are found in or on the glomerular basement membrane. AP activation in C3G is also caused by mutations in complement genes, especially FH, or by autoantibodies (C3 nephritic factor) affecting the C3 convertase (Loirat et al. 2011, Sethi et al. 2012).
Therapeutic Options in Complement Related Diseases
Established therapeutic options in the treatment of aHUS or C3G are limited and include plasmapheresis or substitution with fresh frozen plasma (FFP), immunosuppressive treatment and renal transplantation with a high risk of recurrence of the underlying disease (Braun et al. 2005, Lu et al. 2012, Cataland et al. 2014, Masani et al. 2014). Currently, a number of complement-targeting therapeutics are under investigation and might offer treatment options in the future (Wagner et al. 2010, Ricklin et al. 2013). Among them, eculizumab, a humanized monoclonal anti-05 antibody, blocks TCC formation and has recently been approved for the treatment of aHUS (Zimmerhackl et al. 2010). For C3G, no therapeutic regime has been established yet (Masani et al. 2014). The application of eculizumab in C3G patients led to a partial response in only some patients (Bomback et al. 2012).
Blockade of the late effector functions of complement can be obtained if the cleavage of C5 by the C5 convertase is prevented. The therapeutic monoclonal antibody eculizumab binds human C5 and inhibits its activation into the potent anaphylatoxin C5a and the initiator of the terminal complement pathway C5b by C5 convertase (Parker et al. 2007). Thereby, eculizumab blocks inflammatory signaling and cell lysis by MAC formation, but leaves the C3 convertases and uncontrolled production of C3a unaffected. Eculizumab showed significant improvement in clinical outcome and has been accepted for treatment of complement-mediated diseases including paroxysmal nocturnal hemoglobinuria (PNH) (Hillmen et al. 2006) and aHUS (Zuber et al. 2012).
The central position of C3 in the complement cascade makes it an attractive target for therapeutic interventions, consequently inhibitors acting at the level of C3 have also been designed. Compstatin is a small peptide of 13 amino acids that is being tested pre-clinically (Mastellos et al. 2015). Structural studies have revealed that compstatin binds to the ß-chain of C3 and C3b and blocks the interaction with C3 convertases. Thereby, compstatin inhibits the activation of C3 and also further amplification of the cascade and prevents downstream formation of complement effectors. Compstatin does not prevent cleavage of C3 by proteases such as thrombin nor the “activation” of C3 into C3(H2O) via “Tick-Over” (Ricklin et al. 2015) and has shown efficacy in complement blocking in vitro and in animal models including extracorporeal circulation, sepsis, and PNH. Another approach is the exploitation of our natural panel of RCAs like FH or soluble CR1 or to improve their efficiency by combination of selected domain modules (Ricklin et al. 2013). TT30, containing FH SCR1-5 and CR2 SCR1-4 is designed to accumulate preferentially at sites already under complement-mediated attack. TT30 interacts simultaneously with C3b and C3d merging the functionality of fluid phase FH binding to C3b with CR2 interaction to C3d on the surface of host cells. TT30 showed significant improvement in models of AMD, ischemia/reperfusion injury, and PNH (Merle et al. 2015,). Mini-FH molecules, containing SCR1-4 or SCR 1-5 and SCR19-20 bind C3b and C3d with high affinity and show better efficacy compared to native FH in in vitro models of aHUS and PNH (Hebecker et al. 2013, Schmidt et al. 2013). However, in FH deficient mice, showing a C3G-like phenotype, the therapeutic effects of mini-FH and a murine analog of TT30 on plasma alternative pathway control were comparatively modest, in association with a short half-life (Nichols et al. 2015, Ruseva et al. 2015).
Eculizumab is approved for the application in aHUS and PNH. It blocks the cleavage of C5 by C5 convertase nonspecifically. Patients under eculizumab therapy are at risk for developing severe infectious diseases, especially meningococcal infections like meningitis. On the other hand blockade of TCC formation does not influence C3 convertase activation. This might lead to a sustained production of the anaphylatoxin C3a in treated patients and to continuous deposition of C3 cleavage products in kidneys, as seen in C3G patients under eculizumab therapy. Additionally deposits of the eculizumab antibodies were found in kidney biopsies in eculizumab treated patients (Herlitz et al. 2012). Until now long term effects of theses deposits are not investigated.
Blockade of only C5 convertase might also not be sufficient in all AP related diseases. Single case reports on the effectiveness of eculizumab in C3G patients show only benefits in some patients mostly as a partial remission (Bomback et al. 2012, Legendre et al. 2013).
New complement regulators like compstatin, mini-FH or TT30 try to influence the complement activation on the level of C3 convertase. These developments are under investigation in a preclinical stadium. Eculizumab and other regulators that are currently under development are targeting either C5 convertase or C3 convertase.